Human pancreatic epithelial progenitor cells and methods of isolation and use thereof

ABSTRACT

The invention discloses a substantially pure population of human pancreatic progenitor cells and methods of isolating and culturing the pancreatic progenitor cells. By carefully manipulating the microenvironment of the pancreatic progenitor cells, multiple passages are attainable wherein the pancreatic progenitor cells do not senesce and furthermore, are capable of becoming functional exocrine or endocrine cells. In addition, several methods of use of human pancreatic progenitor cells are disclosed herein.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application is a continuation of U.S. patent application Ser. No.09/546,577, filed on Apr. 10, 2000, now U.S. Pat. No. 6,436,704 theentirety of which is hereby incorporated by reference.

TECHNICAL FIELD

This invention is in the field of developmental biology and cellbiology. Specifically, this invention relates to a population ofpancreatic epithelial progenitor cells that are capable ofdifferentiating into functional endocrine and exocrine cells, methods ofisolating the pancreatic epithelial progenitor cells, characterizationof pancreatic epithelial progenitor cells, and uses of the pancreaticepithelial progenitor cells.

BACKGROUND ART

Stem cell and progenitor cell isolation and characterization are thesubjects of intense research because of the great potential of suchcells. The totipotent stem cells, which have the capacity to become anytype of cell in a human body, give rise to progenitor cells moredifferentiated than the totipotent cell. One of these types ofprogenitor cells is the pre-determined pancreatic epithelial progenitorcell. The pancreatic epithelial progenitor cells have the ability tobecome different types of pancreatic epithelial cells. The differenttypes of pancreatic epithelial cells include acinar cells, islet cells,and ductal cells. Acinar cells are generally found near the head of thepancreas and contain zymogen granules which are readily visible byelectron microscopy. Acinar cells perform exocrine functions bydischarging alkaline digestive juices into the small intestine.Approximately 1500 mL of pancreatic juices are secreted per day andinclude enzymes needed to break up lipids and proteins. Ganong, WilliamF. Review of Medical Physiology, Chapter 26 “Regulation ofGastrointestinal Function”, Fifteenth Edition, Appleton and Lange(1991). There are four types of islet cells, also known as islets ofLangerhans, islet-α, isle-β, islet-δ, and islet-PP. Islet-αcells secreteglucagon which promotes gluconeogenesis, i.e. breakdown of energyreserves to generate more circulating glucose. Islet-β cells secreteinsulin which promotes storage of circulating glucose into accessibleenergy resources. In type I diabetes mellitus, otherwise known asjuvenile diabetes, it is thought that autoimmune attacks on islet-βcells cause defective islet-β cell function, thereby causing a lack ofinsulin to reduce the levels of circulating glucose. Islet-δ cellssecrete somatostatin which regulates the secretion of glucagon andinsulin. The fourth islet cell type islet-PP (pancreatic polypeptide)does not yet have a known function within the pancreas. Another type ofsub-pancreatic cell is the ductal cell. These cells line the ducts thatconnect different parts of the pancreas.

Isolation of pancreatic epithelial progenitor cells, as with other typesof progenitor cells, is difficult because of the ephemeral nature ofprogenitor cells. Manipulation of progenitor cells required forisolation may disturb the fragile progenitor status of these cells andmay cause them to differentiate. Contact with growth factors orsubstrates may also induce a pancreatic progenitor cell to begindifferentiating into exocrine or endocrine cells. Research in the areaof pancreatic cells has resulted in the establishment of severalpancreatic epithelial cell lines derived from rats. Stephan, J. et. al.Endocrinology 140:5841–5854, (1999). Other research includes theisolation of human adult pancreatic cells and the induction of thesepancreatic cells to proliferate into islet-β-like structures withhepatocyte growth factor/scatter factor (HGF/SF). Jeffrey et. al. U.S.Pat. No. 5,888,705. Other research work involves inducing growth ofislet cells from adult pancreatic cells by culturing first inserum-containing, low-glucose medium and then switching to medium withhigher serum and glucose content. WO 9715310. Still other research inthe area of pancreatic progenitor cells includes isolating progenitorcells from pre-diabetic adults and culturing in a serum-containing,pre-defined media that promotes the growth of functional islet cells.U.S. Pat. No. 5,834,308. However, all of these “progenitor” cells giverise only to islet cells. Pancreatic cells of the aforementionedresearch do not have the capacity to differentiate into both endocrineand exocrine cell types. It seems likely that the pancreatic cells ofthe aforementioned research are further committed down thedifferentiation pathway of pancreatic progenitor cells and therefore aredifferent types of pancreatic cells than the human pancreatic progenitorcells of this invention. Furthermore, culturing conditions used in theaforementioned research wherein serum is used to supplement media mayhave adverse consequences. Serum, the fluid portion of blood after bloodhas been allowed to clot, contains many biomolecules such as albumin andα, β, -globulins. In vivo, cells are not normally exposed to anequivalent of serum unless tissue injury was involved. Therefore,culturing pancreatic cells in serum may not accurately reflect thephysiological parameters within which pancreatic cells exist in vivo.

The ideal population of pancreatic progenitor cells should be able todifferentiate into exocrine (i.e. acinar) cells, endocrine (i.e.islet-α, islet-β, islet-δ, and islet-PP) cells as well as ductal cells.Such a population of pancreatic progenitor cells may be useful inclinical settings, for example to treat certain types of diabetes or totreat functionally defective pancreatic cells by transplantation ofpancreatic progenitor cells that can differentiate into functionalpancreatic cells. Accordingly, there is a need for a population ofpancreatic progenitor cells and methods of isolating and culturing thepancreatic progenitor cells such that the differentiation potential ofthe pancreatic progenitor cells is retained while permittingproliferation and avoiding senescence of these cells. The pancreaticprogenitor cells and methods of isolating and culturing these pancreaticprogenitor cells disclosed herein satisfies these needs and alsoprovides related advantages.

DISCLOSURE OF THE INVENTION

This invention is related to the field of developmental and cellbiology. In one aspect, the invention relates to a population ofsubstantially pure human pancreatic epithelial progenitor cells whichhave a pluripotent capability to differentiate into functional exocrineor endocrine pancreatic cells.

In another aspect of this invention, the invention relates to methods ofisolating a population of substantially pure human pancreatic epithelialprogenitor cells which have the pluripotent capability to differentiateinto functional exocrine or endocrine pancreatic cells.

In yet another aspect of this invention, the invention relates tomethods of maintaining a population of substantially pure humanpancreatic epithelial progenitor cells which have the pluripotentcapability to differentiate into functional exocrine or endocrinepancreatic epithelial cells and maintaining or culturing thesepancreatic progenitor cells such that the cells retain their pluripotentcapacity while avoiding senescence.

In still another aspect of this invention, the invention relates tomethods of providing a source of immunogen and the uses of asubstantially pure population of pancreatic progenitor cells as animmunogen.

In still another aspect of this invention, the invention relates tomethods of generating a human pancreatic tissue model using asubstantially pure population of pancreatic progenitor cells as a sourceof pancreatic cells and introducing the pancreatic progenitor cells intoa non-human, mammalian recipient.

In another aspect of this invention, the invention relates to methods ofproviding cell therapy whereby a substantially pure population of humanpancreatic progenitor cells are introduced into a recipient.

In another aspect of this invention, the invention relates to methods ofproviding pharmaceutical drug development wherein a substantially purepopulation of human pancreatic progenitor cells are used as a source ofpancreatic biological components in which one or more of thesepancreatic biological components are the targets of the drugs that arebeing developed.

In another aspect of this invention, the invention relates to methods ofproviding bioassay development wherein a substantially pure populationof human pancreatic progenitor cells are used as a source of nucleicacids or proteins and wherein these nucleic acids or proteins are usedas one or more principal components in a bioassay or the development ofa bioassay.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings(s) will be provided by the Office upon request andpayment of the necessary fee.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows human pancreatic ductal epithelial cells grown in twodifferent types of media. FIG. 1A (left) shows pancreatic epithelialcells grown in CMRL 1066 medium with fibronectin coating on the plate.The large, rounded cells are pancreatic epithelial cells. FIG. 1B(right) shows pancreatic epithelial cells grown in F12/DMEM medium. Thepancreatic epithelial cells have flatten out to form a monolayer.

FIG. 2 shows human pancreatic epithelial cells grown on collagen-coatedplates after three passages. The arrows denote dividing cells.

FIG. 3 shows results of staining of tissue recombinant grafts. FIG. 3Ashows islet formation in the tissue recombinant graft at a magnificationof 20×. FIG. 3B shows islet formation in the tissue recombinant graft ata magnification of 60×. FIG. 3C shows formation of islet, duct, andacinar tissue within the tissue recombinant graft. FIG. 3D shows ductalformation in the tissue recombinant graft. FIG. 3E shows formation ofclusters (or aggregates) of acinar cells in the tissue recombinantgraft.

FIG. 4 shows the results of staining for glucagon (blue) and insulin(brown) in the tissue recombinant graft.

FIG. 5 shows the results of staining for insulin (brown) in the tissuerecombinant graft.

FIG. 6 shows ductal formation in the tissue recombinant.

FIG. 7 shows the results of staining for glucagon (blue) and insulin(brown) in paraffin-embedded tissue section from a tissue graft.

FIG. 8 is a schematic depiction of the development of a pancreatic cellsfrom a totipotent stem cell. The dotted line indicates the stage ofdifferentiation at which the human pancreatic progenitor cell of thisinvention resides.

MODES FOR CARRYING OUT THE INVENTION

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Thisdetailed description should not be construed to limit the presentinvention, as modifications of the embodiments disclosed herein may bemade by those of ordinary skill in the art without departing from thespirit and scope of the present invention. Throughout this disclosure,various publications, patents, and published patent specifications arereferenced by citation. The disclosure of these publications, patents,and published patents are hereby incorporated by reference in theirentirety into the present disclosure.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of immunology, molecular biology,microbiology, cell biology and recombinant DNA, which are within theskill of the art. See, e.g., Sambrook, et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2^(nd) edition (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS INENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J.MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane,eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

Definitions

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used in the specification and claims, the terms “pancreaticepithelial progenitor cells” and “pancreatic progenitor cells” areinterchangeable and refer to “pancreatic epithelial progenitor cells”and “pancreatic progenitor cells” of human origin.

“Pancreatic epithelial progenitor cells” and “pancreatic progenitorcells” refer to dividing progenitor cells found in the pancreas thathave not yet committed to an essentially non-dividing stage of enddifferentiation. “Pancreatic epithelial progenitor cells” and“pancreatic progenitor cells” are derived ultimately from totipotentcells that give rise to pluripotent, tissue-specific cells. Thesepluripotent, tissue-specific, dividing progenitor cells can give rise tocells of the endoderm, ectoderm, or mesoderm. Of the endodermalmultipotent cells, some differentiate into gut-specific, dividingprogenitor cells. Of the gut-specific progenitor cells, some arepre-determined to become pancreatic cells. It is at this stage ofdevelopment that the population of cells claimed herein resides. Morespecifically, the population of “pancreatic epithelial progenitor cells”and “pancreatic progenitor cells” disclosed herein is between the stageat which a gut-specific progenitor cell is pre-determined to become apancreas (or part of a pancreas) and the stage at which apancreas-specific progenitor cell is committed to becoming asub-pancreatic type of cell. Pancreas-specific progenitor cells candifferentiate into several types of cells: acinar, ductal, and islet-α,islet-β, islet-δ, and islet-PP. One exocrine function of the acinarcells is the secretion of digestive juices into the intestine. Oneendocrine function of the islet cells is the secretion of glucagon(islet-α) and insulin (islet-β). The pancreatic progenitor cells of thisinvention have not differentiated into any of the aforementioned typesof sub-pancreatic cells but have the capacity to become any of thesecells.

“Sub-pancreatic” refers to cellular infrastructure within the pancreasas a whole organ. Examples of sub-pancreatic cells include, but are notlimited to, acinar, ductal, and islet cells.

“Totipotent cell” and “totipotent stem cell” are used interchangeablythroughout and refer to a stem cell that has the capacity to become anytype of cell in a mammalian body.

“Pluripotent” and “multipotent” are used interchangeably throughout andrefer to a stage where a cell can still become one of a plurality ofcells but can no longer become any type of cell in the body.“Pluripotent” cells are not referred to as “stem cells” but rather“progenitor cells” because they are progenitors to one or more type of aplurality of cells.

As used herein, “pre-determined pancreatic” refers to a stage ofdevelopment of a multipotent cell that is beyond the stage of beinggut-specific and before the stage of terminally differentiatedpancreatic cells (such as acinar, islet, or ductal cells). Cells whichare “pre-determined pancreatic” are committed to becoming pancreaticcells but have not begun to develop into terminally differentiatedpancreatic cells yet. Different factors cause pre-determined pancreaticcells to begin differentiating. Non-limiting examples include exposureto serum, exposure to insulin growth factor (IGF) or epidermal growthfactor (EGF), contact with surrounding tissue, microenvironment of thecells, and cell-cell contact with surrounding tissue. The chain ofdevelopment begins with a totipotent stem cell which can become any cellin the body. The totipotent stem cell is a true stem cell because of itscellular omnipotency. At any stage beyond the totipotent stem cell,cells become a “pre-determined progenitor” because they have beencommitted down a pathway that no longer enables the cell to become anykind of cell in the body.

An “antibody” is an immunoglobulin molecule capable of binding anantigen. As used herein, the term encompasses not only intactimmunoglobulin molecules, but also anti-idiotypic antibodies, mutants,fragments, fusion proteins, humanized proteins and modifications of theimmunoglobulin molecule that comprise an antigen recognition site of therequired specificity.

The term “antigen” is a molecule which can include one or more epitopesto which an antibody may bind. An antigen is a substance which can haveimmunogenic properties, i.e., induce an immune response. Antigens areconsidered to be a type of immunogen. As used herein, the term “antigen”is intended to mean full length proteins as well as peptide fragmentsthereof containing or comprising one or a plurality of epitopes.

The terms “surface antigens” and “cell surface antigen” are usedinterchangeably herein and refer to the plasma membrane components of acell. These component include, but are not limited to, integral andperipheral membrane proteins, glycoproteins, polysaccharides, lipids,and glycosylphosphatidylinositol (GPI)-linked proteins. An “integralmembrane protein” is a transmembrane protein that extends across thelipid bilayer of the plasma membrane of a cell. A typical integralmembrane protein consists of at least one membrane spanning segment thatgenerally comprises hydrophobic amino acid residues. Peripheral membraneproteins do not extend into the hydrophobic interior of the lipidbilayer and they are bound to the membrane surface by noncovalentinteraction with other membrane proteins. GPI-linked proteins areproteins which are held on the cell surface by a lipid tail which isinserted into the lipid bilayer.

The term “monoclonal antibody” as used herein refers to an antibodycomposition having a substantially homogeneous antibody population. Itis not intended to be limited as regards to the source of the antibodyor the manner in which it is made (e.g. by hybridoma or recombinantsynthesis). Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. In contrast to conventional(polyclonal) antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen.

“A population of monoclonal antibodies” refers to a plurality ofheterogeneous monoclonal antibodies, i.e., individual monoclonalantibodies comprising the population may recognize antigenicdeterminants distinct from each other.

“Immunogen” refers to any substance that induces an immune response. Asubstance that is an immunogen is described as being “immunogenic”.Induction of immune response includes but is not limited to activationof humoral responses (e.g. producing antibodies) or cellular responses(e.g. priming cytotoxic T cells), inflammatory responses (e.g.recruitment of leukocytes), and secretion of cytokines and lymphokines.

The term “heterologous” as applied to a cell used for immunization ortransplantation means that the cell is derived from a genotypicallydistinct entity from the recipient. For example, a heterologous cell maybe derived from a different species or a different individual from thesame species as the recipient. An embryonic cell derived from anindividual of one species is heterologous to an adult of the samespecies.

A cell is of “ectodermal”, “endodermal” or “mesodomal” origin, if thecell is derived, respectively, from one of the three germlayers—ectoderm, the endoderm, or the mesoderm of an embryo. Theectoderm is the outer layer that produces the cells of the epidermis,and the nervous system. The endoderm is the inner layer that producesthe lining of the digestive tube and its associated organs, includingbut not limited to pancreas and liver. The middle layer, mesoderm, givesrise to several organs (including but not limited to heart, kidney, andgonads), connective tissues (e.g., bone, muscles, tendons), and theblood cells.

The terms “medium”, “cell culture medium”, and “culture medium” are usedinterchangeably. The terms refer to the aqueous microenvironment inwhich the mammalian cells are grown in culture. The medium comprises thephysicochemical, nutritional, and hormonal microenvironment.

A cell culture medium is “essentially serum-free” when the percentage byvolume of serum in the medium does not mask antigenic sites or antibodybinding sites on cell surfaces. The term “essentially serum-free”generally applies when the cell culture medium comprises less than about50% serum (by volume), preferably less than about 25% serum, even morepreferably less than about 5% serum, and most preferably less than about0.1% serum.

A cell surface is “substantially free of serum biomolecules” when atleast about 75% of the pancreatic progenitor cell surfaces, morepreferably at least about 90% of the pancreatic progenitor cellsurfaces, even more preferably at least about 95% of the pancreaticprogenitor cell surfaces, and most preferably at least about 99% of thepancreatic progenitor cell surfaces do not have serum biomoleculesderived from serum binding to the cell surface such that antigenic sitesor antibody binding sites are bound or are unavailable for antigenicrecognition by an antibody or a portion of an antibody. Cell surface candetermined by measuring the cell size, either by microscopy or flowcytometry. For example, synthetic beads of various known sizes arecommonly used for calibration in flow cytometry. A small quantity ofcalibrated bead may be mixed with pancreatic progenitor cells and theresultant population is analyzed by flow cytometry. Pancreaticprogenitor cell can then be compared with the size of the calibratedbeads. Calculations of cell surface amount can be accomplished since thesizes of the beads are known.

As used herein, a “substantially pure” population of pancreaticprogenitor cells is a population of cells that is comprised at leastabout 85% pancreatic progenitor cells, preferably at least about 90%,and even more preferably about 95% or more.

A “defined medium” and “basal cell-sustaining medium” are usedinterchangeably herein and refer to a medium comprising nutritional andhormonal requirements necessary for the survival and/or growth of thecells in culture such that the components of the medium are known.Traditionally, the defined medium has been formulated by the addition ofnutritional and growth factors necessary for growth and/or survival.Typically, the defined medium provides at least one component from oneor more of the following categories: a) all essential amino acids, andusually the basic set of twenty amino acids plus cystine; b) an energysource, usually in the form of a carbohydrate such as glucose; c)vitamins and/or other organic compounds required at low concentrations;d) free fatty acids; and e) trace elements, where trace elements aredefined as inorganic compounds or naturally occurring elements that aretypically required at very low concentrations, usually in the micromolarrange. The defined medium may also optionally be supplemented with oneor more components from any of the following categories: a) one or moremitogenic agents; b) salts and buffers as, for example, calcium,magnesium, and phosphate; c) nucleosides and bases such as, for example,adenosine and thymidine, hypoxanthine; and d) protein and tissuehydrolysates.

As used herein, “conditioned media” refers to culture media, free ofintact cells, in which pancreatic epithelial progenitor cells have beengrown. Pancreatic cells grown in nutrient media may release factorswhich promote the continued survival, growth, and maintenance ofpre-existing state of pre-differentiation of the pancreatic progenitorcells. Conditioned media may be used to reconstitute a cell pellet oradded to cells already existing in culture plates. Conditioned media mayalso be used alone or to supplement nutrient media being used to feedpancreatic cells. Since conditioned media derived from nutrient mediaand nutrient media, as described herein, is essential serum-free,conditioned media is also essentially serum-free.

“Standard incubation conditions” refers to the physicochemicalconditions in an incubator designed for tissue culture in which cellsare placed. Generally, the standard incubation conditions are about 37degrees Celsius and about 5% CO₂ content with humidification. All tissueculture techniques and equipment should be performed under sterileconditions. Tissue culture containers refer to any type of containerthat may be used for culturing cells. Non-limiting examples includeflasks and plates.

A “mitogenic agent” or “growth factor” is a molecule which stimulatesmitosis of the mammalian cells. Generally, the mitogenic agent or growthfactor enhances survival and proliferation of mammalian cells in cellculture and is a polypeptide. The mitogenic polypeptide can be a“native” or “native sequence” polypeptide (i.e. having the amino acidsequence of a naturally occurring growth factor) regardless of themethod by which it is produced (e.g. it can be isolated from anendogenous source of the molecule or produced by synthetic techniquesincluding recombinant techniques), or a variant or mutant thereof (seedefinition below). Non-limiting examples include activators of one ormore members of the erbB receptor family; agents which elevate cAMPlevels in the culture medium (e.g. forskolin, cholera toxin, cAMP oranalogues thereof); adhesion molecules such as neural cell adhesionmolecule (N-CAM), laminin or fibronectin; progesterone; neurotrophicfactors such as bone-derived neurotrophic factor (BDNF) and ciliaryneuronotrophic factor (CNTF); neurotrophin-3, -4, -5, or -6;platelet-derived growth factor (PDGF); fibroblast growth factor such asacidic FGF (aFGF) and basic FGF (bFGF); vascular endothelial growthfactor (VEGF); transforming growth factor (TGF) such as TGF-α and TGF-β;insulin-like growth factors, including IGF-I and IGF-II; hormones suchas estrogen, testosterone, thyroid hormone, insulin and any of thosemitogens listed in Table 8.2 at pages 138–139 of Mather, J. P. andRoberts, P. E. (1998) “Introduction to Cell and Tissue Culture”, PlenumPress, New York.

“Pancreatic progenitor cell aggregates”, “pancreatic progenitor cellspheres”, and “pancreatic cell clusters” are used interchangeablythroughout and refers to a mass of a plurality of pancreatic progenitorcells which can form a three-dimensional structure resembling roughly asphere.

A “grafting recombinant”, as used herein, refers to the combined unit ofpancreatic progenitor cell aggregates placed with mesenchymal tissue.Mesenchymal tissue can be of pancreatic or non-pancreatic origin.Mesenchymal tissue can be from a species heterologous to the graftrecipient. Mesenchymal tissue can also be from a species heterologous tothe source of pancreatic progenitor cells. Grafting recombinants can beincubated on substrate, preferably a soft, biological substrate (e.g.agar) for a period ranging from 1 hour to 72 hours, more preferablybetween 6 hours to 24 hours, and even more preferably, overnight with anincubation period of about 8 to 16 hours. Olumi A. F., et. al. CancerResearch 59, 5002–5011, (1999).

“Serum”, as used herein, refers to the fluid phase of mammalian bloodthat remains after blood is allowed to clot.

“Serum biomolecules”, as used herein, refers to biological compositionsfound in serum. Examples include, but are not limited to, albumin,α1-globulin, α2-globulin, β-globulin, and γ-globulin. Serum biomoleculescan include biological compositions, whole or partial, that are eithernaturally found in serum or derived from processing and handling ofserum.

The terms “mammals” or “mammalian” refer to warm blooded vertebrateswhich include but are not limited to humans, mice, rats, rabbits,simians, sport animals, and pets.

Isolation and Maintenance of Pancreatic Progenitor Cells

Pancreatic progenitor cells of this invention are isolated from humanfetal pancreatic tissue. The age of the fetus is between about week 6and about week 40, preferably between about week 8 and about week 26,and even more preferably between about week 12 and about week 22. Thepancreatic tissue can be identified by gross anatomy, outwardappearance, and location within the fetus. Several features of grossanatomy and appearance distinguishing a pancreas are: an elongatedlobulated retroperitoneal gland, lack of capsule, and extension from theconcavity of the duodenum of the intestine to the spleen. The pancreasconsists of a flattened head or caput within the duodenal concavity, anelongated three-sided body extending transversely across the abdomen,and a tail in contact with the spleen. Once identified, fetal pancreatictissue is microdissected. The purpose of microdissection is to separatestructures containing epithelial cells from connective tissue andnon-pancreatic tissue such as fat, membranes, etc. or to separate cellsfrom each other. Non-limiting examples of microdissection includedevices that render mechanical shearing forces (i.e. homogenizer, mortarand pestle, blender, etc.), devices that render cuts or tears (i.e.scalpel, syringes, forceps, etc.), or ultrasonic devices. Alternatively,another method of microdissecting fetal pancreatic tissue is the use ofenzyme treatment. Various enzyme treatments used to microdissect tissueare well known in the art. One method includes the use ofcollagenase-dispase to digest partially sheared pancreatic tissue in abuffered medium that will sustain viability of cells isolated from thepancreatic tissue. A concentration of at least about 0.5 mg/mlcollagenase-dispase is used, more preferably at least about 1 mg/ml andeven more preferably at least about 5 mg/ml. The amount of enzyme willdepend on the age of the fetus and how large the pancreatic tissue is.In the preferred embodiment, pancreatic tissue from fetus between about14 weeks and about 22 weeks is digested with about 5 mg/ml ofcollagenase-dispase. A wide variety of basal cell-sustaining media thatcan be used to keep the pH of the liquid in a range that promotessurvival of pancreatic progenitor cells and to provide additional volumeof liquid within which the enzymatic digestion can occur. Non-limitingexamples include F12/DMEM, Ham's F10 (Sigma), CMRL-1066, Minimalessential medium (MEM, Sigma), RPMI-1640 (Sigma), Dulbecco's ModifiedEagle's Medium (DMEM, Sigma), and Iscove's Modified Eagle's Medium(IMEM). In addition, any of the basal nutrient media described in Hamand Wallace Meth. Enz., 58:44 (1979), Barnes and Sato Anal. Biochem.,102:255 (1980), or Mather, J. P. and Roberts, P. E. “Introduction toCell and Tissue Culture”, Plenum Press, New York (1998) can also beused. Examples of other enzymes that could be used to digest tissueinclude neutral proteases, serine proteases including, but not limitedto, trypsin, chymotrypsin, elastase, collagenase, and thermolysin. Inanother preferred embodiment, enzymes that digest DNA, such as DNAase,are used to cut the DNA into smaller pieces in order to prevent tissueaggregation by free DNA. Treatment of fetal pancreatic tissue withenzyme results in cell yields of various amounts. Some cells are insingle cell suspensions, others are in cell aggregates. Cells notassociated with solid tissue matter can be separated from each other orfrom solid tissue matter or from debris by using a density gradient.Compounds that can be used to create a density gradient include, but arenot limited to, serum (i.e. bovine serum albumin or BSA), ovalbumin,nonionic synthetic polymers of sucrose (i.e. Ficoll™), colloidalpolyvinylpyrrolidone-coated silica (i.e. Percoll™), polyvinylpyrrolidoneor PVP, and methylcellulose. In a preferred embodiment, densitygradients that are capable of neutralizing the enzymes used to digestpancreatic tissues are used. One example of such a density gradient isBSA. The amount of BSA used is about 50% volume-to-volume ratio, morepreferably about 25%, more preferably about 10%, and even morepreferably about 5%. The amount of debris that needs to be removeddepends on several factors, such as the extent of digestion ormechanical shear forces applied to the pancreatic tissue. In some cases,one density gradient is enough to remove debris (e.g. mesenchymaltissue, fatty particles, or broken cell membranes). In other cases, morethan one application of a density gradient will be needed. The desiredproduct is a population of relatively pure pancreatic cell aggregates.

Pancreatic cells are then resuspended in a basal cell-sustaining media.A variety of basal cell-sustaining media is available for use. Examplesinclude, but are not limited to, Ham's F12 medium, RPMI-1640, andCMRL-1066. For more optimal conditions to promote pancreatic progenitorcell survival and growth, a variety of nutrients may be added tosupplement the basal media. Examples include, but are not limited to,insulin, transferrin, epidermal growth factor, ethanolamine,phosphoethanolamine, selenium, triiodothyronine, progesterone,hydrocortisone, forskolin, heregulin, aprotinin, bovine pituitaryextract, and gentamycin. In a preferred embodiment, the followingamounts of nutrients are used to promote pancreatic progenitor cellsurvival and growth: at least about 1 μg/ml insulin and not more thanabout 100 μg/ml insulin, more preferably about 10 μg/ml insulin; atleast about 1 μg/ml transferrin and not more than about 100 μg/mltransferrin, more preferably about 10 μg/ml transferrin; at least about1 ng/ml epidermal growth factor and not more than about 100 ng/mlepidermal growth factor, more preferably about 5 ng/ml epidermal growthfactor; at least about 1×10⁻⁸ M ethanolamine and not more than about1×10⁻² M ethanolamine, more preferably about 1×10⁻⁶ M ethanolamine; atleast about 1×10⁻⁹ M phosphoethanolamine and not more than about 1×10⁻¹M phosphoethanolamine, more preferably about 1×10⁻⁶ Mphosphoethanolamine; at least about 5×10⁻¹² M selenium and not more thanabout 1×10⁻¹ M selenium, more preferably about 2.5×10⁻⁸ M selenium; atleast about 1×10⁻¹⁵ M triiodothyronine and not more than about 5×10⁻¹ Mtriiodothyronine, more preferably about 1×10⁻¹² M triiodothyronine; atleast about 1×10⁻¹³ M progesterone and not more than about 1×10⁻¹ Mprogesterone, more preferably about 1×10⁻⁹ M progesterone; at leastabout 1×10⁻¹⁵ M hydrocortisone and not more than about 1×10⁻¹ Mhydrocortisone, more preferably about 1×10⁻⁹ M hydrocortisone; at leastabout 0.001 μM forskolin and not more than about 50 μM forskolin, morepreferably about 1 μM forskolin; at least about 0.1 nM heregulin and notmore than about 100 nM heregulin, more preferably about 10 nMheregulin,; at least about 1 μg/ml aprotinin and not more than about 100μg/ml aprotinin, more preferably about 25 μg/ml aprotinin; at leastabout 1 μg/ml bovine pituitary extract and not more than about 500 μg/mlbovine pituitary extract, more preferably about 75 μg/ml bovinepituitary extract; at least about 1 μg/ml gentamycin and not more thanabout 1 mg/ml gentamycin, more preferably about 100 μg/ml gentamycin.The pancreatic progenitor cells may be grown on different substrates,depending on the type of physical orientation of the cells desired.Non-limiting examples of substrates that may be used includefibronectin, laminin, collagen, polylysine, nitrocellulose, nylon, andpolytetrafluoroethylene. In one embodiment, pancreatic progenitor cellsare grown on fibronectin-coated tissue culture plates in the preferrednutrient media described above. Pancreatic progenitor cells form cellaggregates when cultured in the preferred nutrient media infibronectin-coated plates. Further, this culturing combination allowsfor separation of undesired mesenchymal cells and pancreatic progenitoraggregates. In a preferred embodiment, purification of pancreatic cellaggregates is readily accomplished by culturing the pancreaticprogenitor cells in preferred media using CMRL 1066 as a basal media ina fibronectin plate. Pancreatic progenitor cells form large, roundclusters of cells that are non-adherent while other cell types (i.e.mesenchymal cells) adhere to the fibronectin coating. The clusters ofpancreatic progenitor cells may then be collected and transferred toanother tissue culture container for subculturing and proliferation.When proliferation of more pancreatic progenitor cell clusters isdesired, the tissue culture container is coated with fibronectin and thepancreatic progenitor cells are cultured in the preferred mediadisclosed herein using CMRL 1066 as a basal media. In anotherembodiment, pancreatic progenitor cells are grown in the preferrednutrient media using F12/DMEM as a basal media in collagen-coated tissueculture containers. Pancreatic progenitor cells form monolayers in thisembodiment.

The frequency of feeding pancreatic progenitor cells may be once a dayor every other day. In one embodiment, pancreatic progenitor cells maybe fed by replacing the entirety of the old nutrient media with newnutrient media. In another embodiment, pancreatic progenitor cells maybe fed with conditioned media in which these cells were grown.Subculturing pancreatic progenitor cells to obtain a greater number ofcells is accomplished by taking pancreatic progenitor cells in clusterform (grown on fibronectin) or in monolayer form (grown on collagen) anddividing the plurality of cells into multiple tissue culture containers.Nutrient media is then added to each of the tissue culture containers toachieve a lower concentration of pancreatic progenitor cells than in theoriginal tissue culture container. The nutrient media that is added isdependent on the type of pancreatic progenitor cell arrangement desired.When monolayer arrangement is desired, then F12/DMEM is used a basalmedia in the preferred nutrient media disclosed herein coupled withcollagen coating in the tissue culture containers. When pancreatic cellclusters are desired, CMRL 1066 is used a basal media in the preferrednutrient media disclosed herein coupled with fibronectin coating in thetissue culture containers. Because the claimed pancreatic progenitorcells are unique to this invention and will secrete factors specific tothese cells, the conditioned media derived from the pancreaticprogenitor cells are also unique. In this invention, pancreaticprogenitor cells form aggregates when grown in the preferred nutrientmedia, defined above, in fibronectin tissue culture plates. When thesubstrate is collagen-coated tissue culture plates, pancreaticprogenitor cells form an attached stromal monolayer. Addition ofconditioned media promotes greater vitality in the pancreatic progenitorcells. A preferred amount of conditioned media is at least about 1% toat least about 25% of total media volume. An even more preferred amountof conditioned media is about 15% of total media volume. A frequency offeeding that is preferable for promoting the survival and growth ofpancreatic progenitor cells is once a week, even more preferable istwice a week, and most preferably every other day. The pancreaticprogenitor cells of this invention can be passaged multiple times whileretaining dividing capability and without inducing differentiation ofthese pancreatic progenitor cells into terminally differentiated acinar,islet, or ductal cells.

Characterization of Pancreatic Progenitor Cells

The population of pancreatic progenitor cells of this invention isolatedin the manner disclosed herein have several defining characteristics.First, the pancreatic progenitor cells are at a stage that can bedescribed as “pre-determined pancreatic”. Of the gut-specific progenitorcells, some are pre-determined to become pancreatic cells. It is at thisstage of development that the population of pancreatic progenitor cellsclaimed herein resides (FIG. 8). The pancreatic progenitor cells of thisinvention have the capacity to become either exocrine or endocrinecells. Endocrine and exocrine cells, as used herein, are defined bytheir secretions. Endocrine cells, such as α-islet cells and β-isletcells secrete glucagon and insulin, respectively. Exocrine cells, suchas acinar cells, secrete a variety of pancreatic digestive juices suchas trypsinogen, α-amylase, and lipases.

Identification of pancreatic progenitor cells may be accomplished bymorphology or specific markers or a combination of both techniques. Asdisclosed herein, pancreatic progenitor cells can be rounded andcyst-like in appearance or elongated in a monolayer formation dependingon the culture conditions in which the pancreatic progenitor cells aregrown. Identification of differentiated pancreatic progenitor cells mayalso be accomplished by morphology. Morphology of islet cells is anovoid shape, about 75 μm to 175 μm in size (long axis). Islet cells tendto be located more towards the tail end of a pancreas (away from theduodenal cavity). Markers that can be used to detect islet cells includebut are not limited to glucagon for islet-α cells, insulin for islet-βcells, somatostatin for islet-γ cells, and pancreatic polypeptide forislet-PP cells. Markers that can be used to detect ductal cells include,but are not limited to, cytokeratins (CK) 7, CK 8, CK 18, CK 19, mucinMUC1, carbonic anhydrase II, and carbohydrate antigen 19.9(sialyl-Lewis-a). Morphology of ductal cells is small, round,approximately 10 μm across the cell, appears to be a tightly packed,cuboidal epithelium. Morphology of acinar cells include a larger sizethan ductal cells, shape, and zymogen granules present within acinarcells. Markers that can be used to identify acinar cells include but arenot limited to carboxypeptidase A and amylase.

Ki67 or PCNA are markers that can be used to determine proliferation ofpancreatic progenitor cells. Pre-determined pancreatic progenitor cellsare still capable of dividing whereas terminally differentiated exocrineor endocrine cells are essentially non-dividing. Staining with Ki67 orPCNA can determine proliferative state of a pancreatic cell underanalysis.

Pancreatic progenitor cells of this invention are maintained at theirpre-existing pre-differentiation state in serum-free media. Basalcell-sustaining media or the preferred nutrient media disclosed hereinor conditioned media may be used to culture the pancreatic progenitorcells in vitro. Different types of substrate on tissue culture platescan be used to obtain either aggregates or monolayers of pancreaticprogenitor cells. The use of fibronectin in conjunction with thepreferred nutrient media disclosed herein results in aggregates ofpancreatic progenitor cells whereas the use of collagen on tissueculture plates results in monolayers of pancreatic progenitor cells.

Pancreatic progenitor cells of this invention have the capacity to bepassaged multiple times in the preferred serum-free nutrient mediadisclosed herein. Multipotency is retained during each passage and atany point after each passage, pancreatic progenitor cells of thisinvention can differentiate into functional exocrine or endocrine cells.In addition, at any point after each passage, pancreatic progenitorcells may be used as an immunogen, for cell therapy, for bioassays, toestablish a human pancreatic model, or for drug discovery and/ordevelopment as disclosed herein.

Another characteristic of the pancreatic progenitor cells of thisinvention is the capacity to differentiate into exocrine or endocrinecells upon transplantation under kidney capsule of a recipient mammal.Prior to transplantation, pancreatic progenitor cells do not makedigestive enzymes, such as amylase or lipase, and will not stainpositive for digestive enzymes. As disclosed herein, pancreaticprogenitor cells can be grown either in pancreatic progenitor cellaggregates or in monolayers and then combined with mesenchymal tissueand placed under a kidney capsule of a recipient mammal. Preferably,human pancreatic progenitor cell aggregates are combined with ratseminal vesicle mesenchymal tissue and placed under the kidney capsuleof a recipient mammal. A portion of the transplant may be removed foranalysis using the markers, morphology, or a combination thereof toidentify the pancreatic cells.

Antibodies, either monoclonal or polyclonal, which can be used toidentify this population of pancreatic progenitor cells include, but arenot limited to, anti-cytokeratin 19, anti-carcinoembryonic antigen(CEA), anti-carbonic anhydrase II, anti-cystic fibrosis transmembraneconductance regulator (CFTR).

Uses of Pancreatic Progenitor Cells

Uses As An Immunogen

A use for pancreatic progenitor cells is as an immunogen. As disclosedin this invention, the unique serum-free culturing conditions allow thecell surfaces of the pancreatic progenitor cells to remain free of serumproteins or serum biomolecules that may bind to the surface. A potentialproblem of antigenic sites that may be “masked” with binding by serumbiomolecules is avoided by using the disclosed serum-free isolation andculturing techniques. Accordingly, a panel of antibodies may begenerated to newly available antigens that were “masked” when usingculture conditions containing serum.

Pancreatic progenitor cells isolated and cultured with the methodsdisclosed herein can be used as an immunogen that is administered to aheterologous recipient. Administration of pancreatic progenitor cells asan immunogen can be accomplished by several methods. Methods ofadministrating pancreatic progenitor cells as immunogens to aheterologous recipient include but are not limited to: immunization,administration to a membrane by direct contact such as swabbing orscratch apparatus, administration to mucous membrane by aerosol, andoral administration. As is well-known in the art, immunization can beeither passive or active immunization. Methods of immunization can occurvia different routes which include but are not limited tointraperitoneal injection, intradermal injection, local injection.Subjects of immunization may include mammals such as mice. The route andschedule of immunization are generally in keeping with established andconventional techniques for antibody stimulation and production. Whilemice are employed in this embodiment, any mammalian subject includinghumans or antibody producing cells therefrom can be manipulatedaccording to the processes of this invention to serve as the basis forproduction of mammalian hybridoma cell lines. Typically, mice areinoculated intraperitoneally or in alternate regions (i.e. footpad, tailbase, etc.) with an immunogenic amount of the pancreatic progenitorcells and then boosted with similar amounts of the immunogen. In analternative, cells grown on non-biological membrane matrix, aresurgically implanted intraperitoneally into the host mammal. Lymphoidcells, preferably spleen lymphoid cells from the mice, are collected afew days after the final boost and a cell suspension is preparedtherefrom for use in the fusion.

Hybridomas are prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C. Nature 256:495–497 (1975) as modified by Buck, D.W., et al., In Vitro, 18:377–381 (1982). Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. The technique involves fusing the myeloma cellsand lymphoid cells using a fusogen such as polyethylene glycol, or byelectrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as HAT medium, to eliminate unhybridizedparent cells. Any of the media described herein can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells areused to produce the monoclonal antibodies of the subject invention. Thehybridomas are expanded and subcloned, if desired, and supernatants areassayed for anti-immunogen activity by conventional immunoassayprocedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescenceimmunoassay).

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen.

In this manner, a panel of novel antibodies to cell surface antigenspecific to a stage of pancreatic progenitor cells can be generatedusing the pancreatic progenitor cells of this invention. Once monoclonalantibodies to cell surface antigens on pancreatic progenitor cells aremade by the method disclosed herein, the antibodies can be used to forseveral uses. The antibodies may be sequenced and cloned for purposes ofgenerating recombinant antibodies or humanized antibodies. Other uses ofpancreatic progenitor cell-specific antibodies include but are notlimited to biological testing or purification (i.e. isolating pancreaticprogenitor cells by methods such as flow cytometry and panning),therapeutic uses (i.e. promoting or arresting cell growth by binding ofantibody to target cell or promoting or arresting growth of a cell massby binding of antibody to target cell), clinical diagnosis, andbiological markers (i.e. identification of other pancreatic ornon-pancreatic cells).

Another use as an immunogen is to modulate overall immune response in aheterologous recipient. As is well-documented in the art, foreignsubstances such as cells or organs introduced into a heterologousrecipient may induce a variety of immune responses. The immune responsescan be in the form of rejection (e.g. in organ transplantation), T cellactivation (e.g. cross-priming), anergy, or tolerance. The overallimmune response can be systemic or localized. In the case where alocalized immune response is desired, for example in the gut region, animmunogen such as pancreatic progenitor cells is introduced into the gutregion in an effective amount. Effective amount can be determined in astepwise fashion in which increasing amounts of pancreatic progenitorcells are introduced into a heterologous recipient and the subsequentimmune response is monitored. Overall immune response (e.g. antibodyproduction, cytokine production, T cell proliferation, anergy,tolerance, etc.) may be monitored by a number of methods including butnot limited to ELISA, proliferation assays, flow cytometry with cellsurface markers, and immunohistochemistry.

Use of Pancreatic Progenitor Cells for Drug Discovery

Another use of pancreatic progenitor cells is related to drug discovery.Since the pre-differentiated multipotent pancreatic progenitor cellpopulation has not been isolated and cultured in the disclosed manner,the pancreatic progenitor cell population may secrete proteins that havenot been heretofore discovered or characterized. Previous culturingtechniques using serum may inhibit the secretion of proteins.Alternatively, proteins may change in function, conformation, oractivity as they are being secreted and interacting with serumbiomolecules. Proteins secreted by pancreatic progenitor cells haveminimal interference from serum biomolecules and thus, may be morephysiologically and topologically accurate. Therefore, proteins secretedby pancreatic progenitor cells may be used as targets for drugdevelopment. In one embodiment, drugs can be made to target specificproteins on pancreatic progenitor cells in vivo. Binding of the drug maypromote differentiation of the pancreatic progenitor cells into specificsub-pancreatic cells, such as islet cells. This approach may be usefulwhen islet cell neogenesis is desired, for example in treatment fordiabetes. In another embodiment, drug specific for regulatory proteinsof pancreatic progenitor cells may be used to arrest growth of aparticular type of cell, for example in cases of cystic fibrosis whereinacinar cells are being replaced by ductal cells. In another embodiment,a drug may be an inhibitor of the growth of stem cells or cancer cellswhich express fetal antigens. Any of these proteins can be used astargets to develop therapeutic antibody, protein, or small moleculedrugs.

Uses of Pancreatic Progenitor Cells for Cell Therapy

In another use, pancreatic progenitor cell lines are used for celltherapy. Transplantation of pancreatic progenitor cells is one suchexample of cell therapy. In cases where different types of pancreaticcells, such as islet cells or acinar cells, are unable to perform theirfunction of secreting insulin or glucagon respectively, transplantationof pancreatic progenitor cells provides a remedy because the pancreaticprogenitor cells of this invention are multipotent and can differentiateinto functional exocrine and endocrine cells. To practice this use,pancreatic progenitor cells are isolated and cultured in serum-free,nutrient-defined media using the methods disclosed. Pancreaticprogenitor cells are grown on fibronectin-coated tissue culture platesto obtain pancreatic progenitor cell aggregates. Pancreatic progenitorcell aggregates are grown under standard incubation conditions for abouthalf a day to about 7 days, more preferably for about 1 day to about 5days, and even more preferably about 3 days. Pancreatic cell aggregatescan then be administered to a recipient and allowed to differentiate. Inan alternative, pancreatic cell aggregates can be used as cellularcarriers of gene therapy wherein pancreatic cells are transfected withone or more genes and enclosed in a delivery device and thenadministered to a recipient. In another embodiment, pancreatic cellaggregates are placed under a kidney capsule and allowed todifferentiate into acinar, ductal, or islet cells. In anotherembodiment, pancreatic cell aggregates are used in a device whichcontains cells and limits access from other cells (i.e. Theracyte®) tolimit immune system responses.

Uses of Pancreatic Progenitor Cells to Make Human Tissue Models

Another use for pancreatic progenitor cells is to create human tissuemodels in non-human mammals. Pancreatic progenitor cell aggregates areplaced on top of mesenchymal tissue to form grafting recombinants. Toform grafting recombinants, about 1 to 15 pancreatic cell spheres, morepreferably about 5 to 8 pancreatic cell spheres, are placed on top ofmesenchymal tissue. The mesenchymal tissue may be either pancreatic ornon-pancreatic tissue and may be derived from a different species fromwhich pancreatic progenitor cells are isolated. In a working example,human pancreatic progenitor cells are placed on top of rat mesenchymalseminal vesicle tissue to form a graft recombinant. A skilled artisanmay determine the optimal combination in a stepwise fashion, by firstisolating human pancreatic progenitor cells using the methods disclosedherein and then combining with mesenchymal tissue from different organs.In some embodiments, a different species, e.g. rat, is used as a sourcefor mesenchymal tissue in combination with human pancreatic progenitorcells. The use of heterologous species allows human-specific markers tobe used to determine the identity of differentiated pancreatic cells.The likelihood of false positives is reduced if rat mesenchymal tissueis used. Likewise, the use of seminal vesicle mesenchymal tissue overpancreatic mesenchymal tissue reduces the likelihood of false positivesin identifying differentiated pancreatic cells. In a preferredembodiment, about 1 to 12 pancreatic progenitor cell spheres, even morepreferably about 5 to 8 pancreatic progenitor cell spheres, are placedon top of rat seminal vesicle mesenchymal tissue. Preferably, about1×10⁴ to about 5×10⁶ mesenchymal cells are used. Even more preferably,about 2×10⁵ to about 5×10⁵ mesenchymal cells are used. A graftrecombinant comprising pancreatic progenitor cell spheres placed onmesenchymal tissue is then placed under the kidney capsule in the fatpad, subcutaneously, or in a device which contains the pancreaticprogenitor cells but limits access of other cells to the pancreaticprogenitor cells (i.e. Theracyte®) in the recipient mammal. Possiblerecipient mammals include but are not limited to mice and rats.Typically in graft situations, donor tissue is vulnerable to attack bythe recipient's immune system. To alleviate graft rejection, severaltechniques may be used. One method is to irradiate the recipient with asub-lethal dose of radiation to destroy immune cells that may attack thegraft. Another method is to give the recipient cyclosporin or other Tcell immunosuppressive drugs. With the use of mice as recipient mammals,a wider variety of methods are possible for alleviating graft rejection.One such method is the use of an immunodeficient mouse (nude or severecombined immunodeficiency or SCID). In a working example, humanpancreatic progenitor cell spheres are placed on rat seminal vesiclemesenchymal tissue and placed under the kidney capsule of animmunodeficient mouse. The graft recombinant remains in the recipientfor about 1 to about 52 weeks, preferably about 5 to about 40 weeks, andeven more preferably about 6 to about 8 weeks before the grafts areharvested and analyzed for pancreatic progenitor cell differentiation.In some cases, a small portion of the graft is needed for analysis.Markers specific for the islet cells (i.e. insulin, glucagon, etc.),ductal cells (i.e. CK 19, etc.), and acinar cells (i.e. amylase, etc.)is utilized in an immunohistochemical analysis. Another set of markersfor exocrine and endocrine functions, such as markers specific forinsulin or glucagon, may also be used to analyze the efficacy of thetransplantation. These markers can be used separately or in combinationwith each other. In addition, a combination of one or more of thesemarkers may be used in combination with cell morphology to determine theefficacy of the transplantation.

In one embodiment, human pancreatic model can be generated in a SCID(severe combined immunodeficiency) mouse. This human pancreatic modelcan be made by utilizing the human pancreatic progenitor cells isolatedand cultured with methods disclosed herein and using the humanpancreatic progenitor cells to make graft recombinants. Graftrecombinants are then placed under the kidney capsule of mice. Afterabout 1 to 10 weeks, preferably about 6 to 8 weeks after implantationunder the kidney capsule, the graft or portion thereof is harvested andanalyzed by immunohistochemistry. Markers specific to exocrine orendocrine function, such as insulin or glucagon are used to analyze theefficacy of the tissue model system. Alternatively, markers specific forpancreatic tissue such as islet cells (i.e. PDX-1), acinar cells (i.e.amylase), ductal cells (i.e. CK 19) are used. Yet another way to assessthe results of pancreatic progenitor cell differentiation is bymorphology. Pancreatic progenitor cells have the appearance of beingsmall and round, about 10 μm across the cell, and in a highly compactedcolumnar epithelium form. Acinar cells have the appearance of largeclusters forming acini. Ductal cells have the appearance of small,round, about 40 μm across the cell, and a compacted, cuboidal columnarepithelium. Islet cells have the appearance of epithelial islandssurrounded by acinar exocrine units. Further, morphology is combinedwith functional markers for insulin and glucagon and cell surfacemarkers for specific cells for a more complete assessment. Therecombinant tissues thus represent a fully human mini-pancreas in amouse. These human pancreatic tissue models can be used to assessefficacy and toxicity of drug candidates being developed to treat type Iand type II diabetes, pancreatitis, pancreatic cancer, and for otherpancreatic insufficiencies. They can also be used to screen any drug forpancreatic toxicity. In a further use, the recipient animal wouldundergo surgical or chemical (i.e. streptazoticin) pancreatic or islet-βcell ablation so the insulin being produced is coming from the graft.

Uses of Pancreatic Progenitor Cells in Bioassays

The pancreatic progenitor cells disclosed herein can be used in variousbioassays. In one use, the pancreatic progenitor cells are used todetermine which biological factors are required for differentiation. Byusing the pancreatic progenitor cells in a stepwise fashion incombination with different biological compounds (such as hormones,specific growth factors, etc.), one or more specific biologicalcompounds can be found to induce differentiation to islet cells.Employing the same stepwise combinations, one or more specificbiological compound can be found to induce differentiation to acinarcells and likewise for ductal cells. Other uses in a bioassay forpancreatic progenitor cells are differential display (i.e. mRNAdifferential display) and protein-protein interactions using secretedproteins from pancreatic progenitor cells. Protein-protein interactionscan be determined with techniques such as yeast two-hybrid system.Proteins from pancreatic progenitor cells can be used to identify otherunknown proteins or other cell types that interact with pancreaticprogenitor cells. These unknown proteins may be one or more of thefollowing: growth factors, hormones, enzymes, transcription factors,translational factors, and tumor suppressors. Bioassays involvingpancreatic progenitor cells and the protein-protein interaction thesecells form and the effects of protein-protein or even cell-cell contactmay be used to determine how surrounding tissue, such as mesenchymaltissue, contributes to pancreatic progenitor cell differentiation.

The following examples provide a detailed description of the isolation,characterization, and use of pancreatic progenitor cells. These examplesare not intended to limit the invention in any way.

EXAMPLES Example 1 Isolation of Pancreatic Progenitor Cells

Fetal pancreas (gestational age 14–22 weeks) was mechanically pulledapart by microdissection under a stereo microscope prior to enzymaticdissociation. Enzyme treatment consisted of placing the partlydissociated tissue in 1 ml F12/DMEM medium containing 5 mg/mlcollagenase-dispase, 20 μg/ml soybean trypsin inhibitor and 50 μg/mlDNAase for 15 minutes at 37 degrees Celsius.

Cell aggregates were layered on top of a 5% (by volume) BSA gradient andwashed by centrifugation for 6 minutes at 900 rpm. Pelleted cells whichwere still in aggregate form were resuspended in growth mediumconsisting of CMRL 1066 nutrient medium containing the followingfactors:

Insulin 10 μg/ml Transferrin 10 μg/ml Epidermal growth factor 5 ng/mlEthanolamine 10⁻⁶ M Phosphoethanolamine 10⁻⁶ M Selenium 2.5 × 10⁻⁸ MTriiodothyronine 10⁻¹² M Progesterone 10⁻⁹ M Hydrocortisone 10⁻⁹ MForskolin 1 μM Heregulin 10 nM Aprotinin 25 μg/ml Bovine pituitaryextract 75 μg/ml Gentamycin 100 μg/ml

Resuspended cell aggregates were aliquoted into fibronectin-coated wells(6–12) of a 24-well dish and incubated at 37 degrees Celsius in ahumidified 5% Co₂ incubator for 72 hours. After 72 hours, the epithelialcells formed suspended spherical structures (FIG. 1A) and themesenchymal or stromal cells were attached to the surface of the well.When monolayer formation was desired, the pancreatic aggregates orpancreatic spheres from 6 of the wells were collected with a micropipetand placed on a collagen-coated 60 mm dish using F12/DMEM as basalnutrient media with the nutrients supplements as disclosed. Within 24hours, the structures attached and the cells from the structure spreadout onto the collagen to form an epithelial monolayer (FIG. 1B). Thesepancreatic progenitor cells could be passaged at least three times (FIG.2).

Example 2 Use of Pancreatic Progenitor Cells in Transplants

For the purpose of recombinant grafting, the cells were left in thespherical state from the time of original plating or the monolayers werereleased from the collagen and grown in non-coated flasks where theyremained in suspension and re-aggregated into spherical structures.

For the purpose of grafting, the spheres were placed on top of seminalvesicle mesenchyme from e15 rats, usually 5–8 spheres to a mesenchymeaggregate of 2×10⁵ to 5×10⁵ cells. Each recombinant was placed on agarand incubated overnight at 37 degrees in a 5% CO₂ humidified chamber.

The grafting consisted of placing from 3–6 recombinants under the kidneycapsule of an immunodeficient mouse (nude or SCID) and left for 6–8weeks. The grafts were then harvested and processed forimmunohistochemistry.

The result of pancreatic tissue recombinant graft transplantation wasassessed by morphology. Pancreatic progenitor cells have the appearanceof being small and round, about 10 μm across the cell, and in a highlycompacted columnar epithelium form. Acinar cells have the appearance oflarge clusters forming acini (FIG. 3E). Ductal cells have the appearanceof small, round, about 40 μm across the cell, and a compacted, cuboidalcolumnar epithelium (FIG. 3D). Islet cells have the appearance ofepithelial islands surrounded by acinar exocrine units (FIGS. 3A, 3B,3C, and FIG. 7).

Example 3 Determining the Identity of Transplanted Pancreatic ProgenitorGraft Cells, Differentiation State of Pancreatic Progenitor Cells, andTheir Function

After the pancreatic spheres have been transplanted under the kidneycapsule of mice and allowed to remain at that location for 6–8 weeks,the grafts were harvested and analyzed for identity of pancreatic cellsby immunohistochemistry and function. The grafts have been shown toexpress insulin and glucagon (FIGS. 4, 5, and 7). Furthermore, thetissue graft recombinants have shown the formation of ductal structures(FIG. 6). Therefore, the tissue recombinant grafts yielded functionalpancreatic cells that could express insulin and glucagon and form ductalstructures.

1. A method of providing cell therapy to a human recipient lacking orhaving functionally defective pancreatic epithelial cells, comprisingadministering to the human recipient an isolated, substantially purepopulation of human pancreatic progenitor cells that are capable ofdifferentiating into acinar, ductal, and islet cells, wherein saidadministration is made to the pancreas of the human recipient.
 2. Themethod of claim 1 further comprising the step of immunosuppressing therecipient.
 3. The method of claim 1 further comprising the step ofproviding said pancreatic progenitor cells in a barrier device thatlimits the immune system response of the recipient against saidpancreatic progenitor cells.
 4. The method of claim 1 wherein thepancreatic progenitor cells are grown in serum-free media beforeadministering to the human recipient.
 5. The method of claim 1comprising an additional step of determining the level of therecipient's pancreatic cell function subsequent to said administration.6. The method of claim 5 wherein the step of determining the level ofsaid recipient's pancreatic cell function comprises measuring insulinlevel in the recipient.
 7. The method of claim 5 wherein the step ofdetermining the level of said recipient's pancreatic cell functioncomprises measuring glucagon level in the recipient.
 8. The method ofclaim 1 wherein the human recipient has diabetes.
 9. The method of claim1 wherein the human recipient lacks or has functionally defectivepancreatic acinar, ductal or islet cells.
 10. A method of providing celltherapy to a human recipient lacking or having functionally defectivepancreatic epithelial cells, comprising administering to the humanrecipient an isolated, substantially pure population of human pancreaticprogenitor cells that are capable of differentiating into acinar, ductaland islet cells, wherein said administration is made to the kidneycapsule of the human recipient.
 11. The method of claim 10 comprisingthe step of immunosuppressing the patient.
 12. The method of claim 10further comprising the step of providing said pancreatic progenitorcells in a barrier device that limits the immune system response of therecipient against said pancreatic progenitor cells.
 13. The method ofclaim 10 wherein the pancreatic progenitor cells are grown in serum freemedia before administering to the human recipient.
 14. The method ofclaim 10 comprising an additional step of determining the level of therecipient's pancreatic cell function subsequent to said administration.15. The method of claim 14 wherein the step of determining the level ofsaid recipient's pancreatic cell function comprises measuring insulinlevel in the recipient.
 16. The method of claim 14 wherein the step ofdetermining the level of said recipient's pancreatic cell functioncomprises measuring glucagon level in the recipient.
 17. The method ofclaim 10 wherein the human recipient has diabetes.
 18. The method ofclaim 10 wherein the human recipient lacks or has functionally defectivepancreatic acinar, ductal or islet cells.